水蛭素在噬菌体表面的展示

Hirudin Display on the Surface of Bacteriophage M13 *

水蛭素是凝血酶强有力的天然抑制剂。通过改造噬菌质粒并构建水蛭素表达载体ｐＣＡＮＴＡＢ５Ｇ８Ｈｉｒ，使水蛭素基因通过接头与噬菌体Ｍ１３的ｇｐ３（１９７～４０６）基因片段融合。表达产物在ｇｐ８信号肽的引导下到达大肠杆菌周质，在辅助噬菌体Ｍ１３ＫＯ７的帮助下组装到丝状噬菌体外壳上。展示在噬菌体表面的水蛭素仍然具有与凝血酶结合并抑制酶活性的作用，说明展示的水蛭素保持了正确的空间构象和生物学活性。水蛭素在噬菌体表面的功成展示为进一步开展其实验定向进化以及结构与功能关系的研究打下基础。

Hirudin was fused to the N terminus of M13 minor protein gp3(197～406) through a linker GGGS by inserting both the hirudin gene and the gp8 signal sequence into the modified phagemid vector pCANTAB 5V to construct pCANTAB 5G8 Hir.The expressed fusion protein was directed by gp8 signal peptide into the periplasm and assembled to the phage particle to form the hirudin phage.The fusion protein and fusion phage were detected with biotin thrombin by Westernblotting analysis.Antithrombin activity analysis confirmed that the fusion protein and fusion phage bear the similar conformation to the native hirudin.The successful display of hirudin on the surface of M13 phage laid a sound foundation for the further study on directed evolution of antithrombotic proteins with altered properties.